scholarly journals Stable isotope dimethyl labelling for quantitative proteomics and beyond

2016 ◽  
Vol 374 (2079) ◽  
pp. 20150364 ◽  
Author(s):  
Jue-Liang Hsu ◽  
Shu-Hui Chen
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Quantitative Proteomics ◽  
Large Scale ◽  
Protein Identification ◽  
Cost Effective ◽  
Quantitative Mass Spectrometry ◽  
Post Translational Modifications ◽  
Labelling Method ◽  
Identification And Characterization

Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’.

Mass Spectrometry Techniques: Principles and Practices for Quantitative Proteomics

2020 ◽  
Vol 21 ◽  
Author(s):  
Rocco J. Rotello ◽  
Timothy D. Veenstra
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Protein Identification ◽  
Dynamic Nature ◽  
Complete Coverage ◽  
The Past ◽  
Proteome Level ◽  
A Cell ◽  
Quantitative Changes ◽  
Made In

: In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and metabolites present within a cell. While genomics has led to a dramatic increase in our understanding of such things as disease morphology and how organisms respond to medications, it is critical to obtain information at the proteome level since proteins carry out most of the functions within the cell. The primary tool for obtaining proteome-wide information on proteins within the cell is mass spectrometry (MS). While it has historically been associated with the protein identification, developments over the past couple of decades have made MS a robust technology for protein quantitation as well. Identifying quantitative changes in proteomes is complicated by its dynamic nature and the inability of any technique to guarantee complete coverage of every protein within a proteome sample. Fortunately, the combined development of sample preparation and MS methods have made it capable to quantitatively compare many thousands of proteins obtained from cells and organisms.

Data Mining in Protein Identification by Tandem Mass Spectrometry

2011 ◽  
pp. 472-478
Author(s):  
Haipeng Wang
Keyword(s):  
Mass Spectrometry ◽  
Data Mining ◽  
Tandem Mass Spectrometry ◽  
Large Scale ◽  
Protein Identification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
Fragmentation Pattern ◽  
Post Translational Modification ◽  
Challenges And Opportunities

Protein identification (sequencing) by tandem mass spectrometry is a fundamental technique for proteomics which studies structures and functions of proteins in large scale and acts as a complement to genomics. Analysis and interpretation of vast amounts of spectral data generated in proteomics experiments present unprecedented challenges and opportunities for data mining in areas such as data preprocessing, peptide-spectrum matching, results validation, peptide fragmentation pattern discovery and modeling, and post-translational modification (PTM) analysis. This article introduces the basic concepts and terms of protein identification and briefly reviews the state-of-the-art relevant data mining applications. It also outlines challenges and future potential hot spots in this field.

scholarly journals Stable Isotope Labeling ofArabidopsis thalianaCells and Quantitative Proteomics by Mass Spectrometry

2005 ◽  
Vol 4 (11) ◽  
pp. 1697-1709 ◽  
Author(s):  
Albrecht Gruhler ◽  
Waltraud X. Schulze ◽  
Rune Matthiesen ◽  
Matthias Mann ◽  
Ole N. Jensen
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Quantitative Proteomics ◽  
Isotope Labeling ◽  
Stable Isotope Labeling

scholarly journals In vivo measurement of synthesis rate of multiple plasma proteins in humans

2006 ◽  
Vol 291 (1) ◽  
pp. E190-E197 ◽  
Author(s):  
Abdul Jaleel ◽  
Vandana Nehra ◽  
Xuan-Mai T. Persson ◽  
Yves Boirie ◽  
Maureen Bigelow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Synthesis ◽  
Stable Isotope ◽  
Plasma Proteins ◽  
Large Scale ◽  
Protein Quantification ◽  
Gas Chromatography Mass Spectrometry ◽  
Synthesis Rate ◽  
Wide Range

Advances in quantitative proteomics have facilitated the measurement of large-scale protein quantification, which represents net changes in protein synthesis and breakdown. However, measuring the rate of protein synthesis is the only way to determine the translational rate of gene transcripts. Here, we report a technique to measure the rate of incorporation of amino acids from ingested protein labeled with stable isotope into individual plasma proteins. This approach involves three steps: 1) production of stable isotope-labeled milk whey protein, oral administration of this intrinsically labeled protein, and subsequent collection of blood samples; 2) fractionation of the plasma and separation of the individual plasma proteins by a combination of anion exchange high-pressure liquid chromatography and gel electrophoresis; and 3) identification of individual plasma proteins by tandem mass spectrometry and measurement of stable isotopic enrichment of these proteins by gas chromatography-mass spectrometry. This method allowed the measurement of the fractional synthesis rate (FSR) of 29 different plasma proteins by using the same precursor pool. We noted a 30-fold difference in FSR of different plasma proteins with a wide range of physiological functions. This approach offers a tremendous opportunity to study the regulation of plasma proteins in humans in many physiological and pathological states.

scholarly journals Peptide and protein analysis with mass spectrometry

2002 ◽  
Vol 16 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Sunia A. Trauger ◽  
William Webb ◽  
Gary Siuzdak
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Protein Identification ◽  
Data Interpretation ◽  
Maldi Mass Spectrometry ◽  
Protein Characterization ◽  
Accurate Information ◽  
Molecular Weight Determination ◽  
Peptide Fragments ◽  
Identification And Characterization

Mass spectrometry (MS) is rapidly becoming a fundamental tool for biologists and biochemists in their efforts to characterize cellular function. Recent advancements in MS technology and front-end methodologies, along with the completion of the human genome have greatly popularized its use by researchers for protein identification and characterization. This paper is a general overview of how mass spectrometry is being used for the analysis of peptides and proteins, focusing on its application to molecular weight determination. Sample preparatory and cleanup techniques used in our laboratory for protein and peptide analysis are provided, along with a discussion of data interpretation. The utility of mass spectrometry for protein and peptide analyses lies in its ability to provide highly accurate molecular weight information on intact molecules. The ability to generate such accurate information can be extremely useful for protein identification and characterization. For example, a protein can often be unambiguously identified by the accurate mass analysis of its constituent peptides produced by either chemical or enzymatic treatment of the sample. Furthermore, protein identification can also be facilitated by analysis of the protein's proteolytic peptide fragments in the gas phase; fragment ions generated inside the mass spectrometer via collision-induced dissociation (CID) to yield information about the primary structure and modifications. This overview describes how electrospray ionization (ESI) and matrix‒assisted laser desorption/ionization (MALDI) mass spectrometry is being used for peptide and protein characterization focusing on its application to molecular weight determination.

N-terminal Cyclization of Peptides in Large-scale Protein Identification Based on Biological Mass Spectrometry

2009 ◽  
Vol 37 (7) ◽  
pp. 950-954
Author(s):  
Zhuang LU ◽  
Li-Yan ZHAO ◽  
Yang-Jun ZHANG ◽  
Yun CAI ◽  
Yu-Lin DENG ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Protein Identification ◽  
Biological Mass Spectrometry

scholarly journals Proteomics of protein trafficking by in vivo tissue-specific labeling

2020 ◽  
Author(s):  
Ilia A. Droujinine ◽  
Dan Wang ◽  
Yanhui Hu ◽  
Namrata D. Udeshi ◽  
Luye Mu ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Large Scale ◽  
Fat Body ◽  
Secreted Proteins ◽  
Intracellular Protein ◽  
Quantitative Mass Spectrometry ◽  
Subcellular Compartment ◽  
Human Orthologs ◽  
Identification And Characterization

AbstractSecreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors’ large-scale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors. This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states.One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteome-wide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles.

scholarly journals Quantitative Proteomics in Drosophila with Holidic Stable-Isotope Labeling of Amino Acids in Fruit Flies (SILAF)

2020 ◽  
pp. 75-87
Author(s):  
Florian A. Schober ◽  
Ilian Atanassov ◽  
Christoph Freyer ◽  
Anna Wredenberg
Keyword(s):  
Amino Acids ◽  
Stable Isotope ◽  
Posttranslational Modifications ◽  
Quantitative Proteomics ◽  
Isotope Labeling ◽  
Cost Effective ◽  
Fruit Flies ◽  
Stable Isotope Labeling ◽  
Functional Studies ◽  
Wet Lab

AbstractProtein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.

scholarly journals Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

2021 ◽  
Author(s):  
Timothy J Aballo ◽  
David S Roberts ◽  
Jake A Melby ◽  
Kevin M Buck ◽  
Kyle A Brown ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Protein Identification ◽  
Heart Tissue ◽  
Tryptic Digestion ◽  
Bottom Up ◽  
Preparation Methods ◽  
Liquid Chromatography Tandem Mass ◽  
And Function ◽  
Identification And Quantification

Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample clean-up to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4,000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.

Sign in / Sign up

Export Citation Format

Share Document